Not Applicable
The present invention relates generally to recombinant avian interferon polypeptides and genetic sequences encoding same, and uses therefor. In particular, the present invention is directed to the use of recombinant avian IFN-xcex3 polypeptides as an immune response modulator and as a growth enhancing agent in avian species. The present invention is particularly useful in the prophylactic and therapeutic treatment of birds against coccidiosis and the causative agent thereof.
1. Field of the Invention
The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research into the medical and veterinary fields. Cytokine research is of particular importance, especially as these molecules regulate the proliferation, differentiation and function of a great variety of cells, such as cells involved in mediating an immune response. Administration of recombinant cytokines or regulating cytokine function and/or synthesis is becoming, increasingly, the focus of medical research into the treatment of a range of disease conditions in humans and animals. The present invention seeks to provide novel reagents and methods that employ recombinant cytokine polypeptides, for the treatment of disease conditions in birds.
2. Description of Related Art Including Information Disclosed Under 37 C.F.R. 1.97 and 1.98
In mammals, interferons (IFN) represent a family of cytokines that share the capacity to inhibit viral replication and to exert effects on immune function. There are two distinct types of IFN. Type I IFN is produced by a variety of cell types in response to viral infection and includes IFN-xcex1 and -xcex2. Typically, IFN-xcex1 is produced by leukocytes such as monocytes and macrophages while fibroblasts and epithelial cells are the major source of IFN-xcex2. Type I IFNs share a high degree of amino acid homology, bind to the same cell surface receptor and there biological functions are resistant to heat and low pH treatment. (Weissmann and Weber, 1986).
In contrast, the production of IFN-xcex3 in mammals is restricted to activated T cells and NK cells and is encoded by a gene that is unrelated to those which express IFN-xcex1 or IFN-xcex2. Features that distinguish IFN-xcex3 from -xcex1/xcex2 include their binding to different cell surface receptors and that the former is exquisitely sensitive to heat and low pH treatment (Weissmann and Weber, 1986). Another distinction is the ability of IFN-xcex3, but not IFN-xcex1 or IFN-xcex2, to stimulate macrophages to produce reactive nitrogen intermediates such as nitric oxide, nitrate and nitrite (Fast et al, 1993; Huang et al, 1993).
Chicken T cells produce IFN following stimulation with antigen or mitogen (Prowse and Pallister, 1989; Lowenthal et al, 1993; Pusztai et al, 1986; Weiler and von Bulow, 1987; Dijkmans et al, 1990) as measured by the ability to protect chick embryonic fibroblasts (CEF) from virus-mediated lysis. There has been controversy as to whether this IFN activity is the Type I interferon, or alternatively, an avian equivalent of mammalian IFN-xcex3 (Lillehoj et al, 1992).
The gene for chicken Type I IFN (ChIFN-xcex1) has recently been cloned (Sekellick et al, 1994) and when the protein was compared to mammalian IFNs it was shown to have 20-24% amino acid sequence identity to Type I IFNs, whereas it was unrelated to known mammalian IFN-xcex3 polypeptides. Furthermore, recombinant ChIFN-xcex1 was shown to have antiviral activity, but lacked macrophage activating function in that it was unable to induce nitrite secretion in monocytes (Schultz et al, 1995), consistent with the properties of mammalian Type I IFN.
U.S. Ser. No. 08/765,381, which is incorporated herein by way of reference, teaches genetic sequences encoding avian IFN-xcex3 from chicken reticuloendotheliosis virus (REV)-transformed spleen cell cultures (Lowenthal et al, 1995 a, b). That specification further teaches the isolation of homologous IFN-xcex3-encoding genetic sequences from species other than chickens, gene constructs and viral vectors comprising said genetic sequences, methods for the production of recombinant avian IFN-xcex3 using the said gene constructs and viral vectors, and methods of prophylaxis and treatment using the recombinant polypeptides. The avian and mammalian IFN-xcex3 polypeptides are only 30% identical, as determined using the ClustalW programme and the algorithm of Thompson et al., 1994), or alternatively, 32% identical as determined using the ClustalV programme.
Birds suffer from a variety of diseases that represent a considerable cost to the poultry industry, including diseases that are produced by bacteria and viruses, such as, for example, infectious bronchitis virus, avian infectious laryngeotracheitis virus, infectious bronchitis virus, Newcastle disease virus, Marek""s Disease virus, chicken anemia virus, avian influenza virus, E. coli, Salmonella ssp., Eimeria ssp. and Mycoplasma ssp., amongst others.
For example, the causative agent of coccidiosis in birds, Eimeria ssp., in particular E. acervulina, is capable of infecting birds at any period in their life cycle. Coccidiosis represents a considerable cost in terms of reduced productivity, because the disease in birds prevents digestion for about 4-5 days, thereby leading to a considerable weight loss, costing the industry approximately $700 million per annum. Current treatments for coccidiosis include the use of chemicals in feed stocks.
This application is a continuation-in-part application of U.S. Ser. No. 08/765,381, the entire contents of which are incorporated herein by way of reference.
Bibliographic details of the publications referred to in this specification by author are collected at the end of the description.
Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification appear after the claims.
Throughout this specification and the claims that follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d will be understood to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. the invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
In work leading up to the present invention, the inventors sought to produce improved methods for the prophylactic and therapeutic treatment of a wide range of diseases in birds, and, in particular, the prophylactic and therapeutic treatment of coccidiosis.
The inventors cloned avian IFN-xcex3 cDNAs and genes and produced recombinant gene constructs comprising same for the expression of recombinant IFN-xcex3 polypeptides therefrom, as described in U.S. Ser. No. 08/765,381 which is incorporated herein by way of reference.
Immunoreactive molecules, in particular polyclonal and monoclonal antibodies have been produced to the recombinant avian IFN-xcex3 polypeptide of the invention.
Additionally, the inventors have discovered that avian IFN-xcex3 administered to birds is capable of promoting the growth of birds, as well as preventing weight loss associated with pathogenic infections when administered thereto as a prophylactic or therapeutic reagent by any means. The present invention clearly encompasses the use of avian IFN-xcex3 as an immunomodulatory molecule both in homologous species and across species boundaries.
Accordingly, one aspect of the present invention provides a method of treatment or prophylaxis of birds exposed to or infected with a pathogenic organism, said method comprising administering thereto an immunoresponsive effective amount of an avian IFN-xcex3 cytokine polypeptide for a time and under conditions sufficient to maintain, stimulate or enhance the immmunoresponsiveness of said bird, wherein said avian IFN-xcex3 cytokine polypeptide is selected from the group consisting of:
(a) a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 2-7;
(b) a polypeptide having the amino acid sequence set forth as the mature protein region of any one of SEQ ID NOs: 2-7;
(c) a polypeptide encoded by DNA present in an avian DNA library, wherein said DNA hybridizes under conditions of at least moderate stringency with a probe having a sequence complementary to at least 50 contiguous nucleotides of SEQ ID NO: 1;
(d) a polypeptide encoded by a nucleotide sequence that is degenerate with a DNA molecule according to (c); and
(e) a polypeptide comprising at least 10 contiguous amino acids of any one of SEQ ID NOs: 2-7, wherein said polypeptide has immunomodulatory activity.
Because of the maintenance, stimulation or enhancement of the immmunoresponsiveness of birds administered with the subject cytokine polypeptide, this aspect of the invention relates further to the enhancement and/or stimulation of an immune response to one or more antigens in a bird, wherein an immunomodulatingly effective amount of an avian IFN-xcex3 cytokine polypeptide is optionally administered with an antigen or pathogenic agent against which an immune response is desired. In such an embodiment of the invention, the administered avian IFN-xcex3 cytokine polypeptide acts essentially as an adjuvant, such as, for example, may be used in a vaccine composition.
A second aspect of the invention provides a method of enhancing the growth performance of a healthy or diseased bird, said method comprising administering to said bird an avian IFN-xcex3 cytokine polypeptide for a time and under conditions sufficient to induce weight gain in said healthy or diseased bird or to prevent weight loss in said diseased bird, wherein said avian IFN-xcex3 cytokine polypeptide is selected from the group consisting of:
(a) a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 2-7;
(b) a polypeptide having the amino acid sequence set forth as the mature protein region of any one of SEQ ID NOs: 2-7;
(c) a polypeptide encoded by DNA present in an avian DNA library, wherein said DNA hybridizes under conditions of at least moderate stringency with a probe having a sequence complementary to at least 50 contiguous nucleotides of SEQ ID NO: 1;
(d) a polypeptide encoded by a nucleotide sequence that is degenerate with a DNA molecule according to (c); and
(e) a polypeptide comprising at least 10 contiguous amino acids of any one of SEQ ID NOs: 2-7, wherein said polypeptide has immunomodulatory activity.
This aspect of the invention particularly relates to the prevention of weight loss associated with coccidiosis of birds, wherein said avian IFN-xcex3 cytokine polypeptide is administered to a bird selected from the group consisting of:
(i) healthy birds that are susceptible to infection by Eimeria spp.;
(ii) asymptomatic birds infected with Eimeria spp.; and
(iii) birds suffering from coccidiosis.
According to this embodiment, it is preferred to administer a composition comprising the avian IFN-xcex3 cytokine polypeptide for a time and under conditions sufficient to induce weight gain in said healthy or infected bird or to prevent weight loss in said infected bird.
The present invention clearly extends to compositions for use in performing the inventive methods supra, such as, for example, those compositions of matter comprising avian IFN-xcex3 cytokine polypeptides and antibodies capable of binding thereto in combination with one or more suitable carriers and/or excipients and/or diluents, in particular those carriers and/or excipients and/or diluents suitable for veterinary use.
Accordingly, a third aspect of the invention provides an antibody molecule that is capable of binding to an avian IFN-xcex3 cytokine polypeptide as described supra, in particular antibodies that bind to chicken IFN-xcex3.
A fourth aspect of the invention provides a composition for enhancing the growth performance of a bird comprising a carrier, excipient or diluent in combination with an amount of a recombinant avian IFN-xcex3 cytokine polypeptide selected from the group consisting of:
(a) a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 2-7;
(b) a polypeptide having the amino acid sequence set forth as the mature protein region of any one of SEQ ID NOs: 2-7;
(c) a polypeptide encoded by DNA present in an avian DNA library, wherein said DNA hybridizes under conditions of at least moderate stringency with a probe having a sequence complementary to at least 50 contiguous nucleotides of SEQ ID NO: 1;
(d) a polypeptide encoded by a nucleotide sequence that is degenerate with a DNA molecule according to (c); and
(e) a polypeptide comprising at least 10 contiguous amino acids of any one of SEQ ID NOs: 2-7, wherein said polypeptide has immunomodulatory activity.
The invention also provides a vaccine composition for the prophylactic treatment of a bird against a pathogenic organism comprising an antigen in combination with an amount of a recombinant avian IFN-xcex3 cytokine polypeptide selected from the group consisting of:
(a) a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 2-7;
(b) a polypeptide having the amino acid sequence set forth as the mature protein region of any one of SEQ ID NOs: 2-7;
(c) a polypeptide encoded by DNA present in an avian DNA library, wherein said DNA hybridizes under conditions of at least moderate stringency with a probe having a sequence complementary to at least 50 contiguous nucleotides of SEQ ID NO: 1;
(d) a polypeptide encoded by a nucleotide sequence that is degenerate with a DNA molecule according to (c); and
(e) a polypeptide comprising at least 10 contiguous amino acids of any one of SEQ ID NOs: 2-7, wherein said polypeptide has immunomodulatory activity.
In particularly preferred embodiments of the present invention, the inventive compositions described herein utilize recombinant chicken IFN-xcex3, or cells infected with recombinant FAV expressing chicken IFN-xcex3, or alternatively, recombinant FAV expressing chicken IFN-xcex3, or nucleic acid encoding chicken IFN-xcex3.